The transmission electron microscope (TEM) has been used for years to study biological systems down to angstrom level resolution but typical samples are prepared using chemical fixation, staining, and dehydration which can introduce artifacts. Vitrification of samples (rapidly cooling liquid into an amorphous solid phase) preserves the delicate spatial organization of hydrated systems without the damage produced when crystalline water is formed. It is well known that vitrified prokaryotic cells can be studied using the transmission electron microscope but the technique is limited to specimens with a maximum thickness of 0.5 to 1 micrometers. Even with the ability to study such thick samples, the resolution is greatly diminished by the increased interaction volume of the beam. Cryo-ultramicrotomy is a possible technique for preparing thin TEM samples regardless of cell size, but samples prepared in this manner often become distorted and deformed by the cutting process. Artifacts include compression of features of up to 50% in the cutting direction. Additionally, cryo-ultramicrotomy does not provide a method for choosing a site of interest for preparation and often many attempts are required before a useful sample is prepared.
FIBSEM sample preparation for later analysis in a transmission electron microscope (TEM) or scanning transmission electron microscope (STEM) has become the preferred technique in the field of electron microscopy. Using the TEM or STEM requires an electron transparent section which usually means the sample is less than 100 nanometers thick for high resolution purposes. The combination of a nanomanipulator, SEM and FIB in one chamber allows site-specific sample preparation and has proven to be valuable to material science and semiconductor research and failure analysis. This technique, however, is currently not available to those who wish to study hydrated systems such as biological samples or those who have samples which are reactive under charged particle beams such as polymers and other unstable films. Cryogenically cooled samples will not maintain cold temperatures necessary for preservation when in contact with an ambient temperature probe.
Some commercially available nanomanipulators have a rod which runs from the outside to the inside of the chamber. Other manipulators are mounted inside the vacuum chamber. Existing nanomanipulator probes cannot be used with hydrated systems such as biological samples or those who have samples which are reactive under charged particle beams such as polymers and other unstable films.
For these and other reasons, FIBSEM in-situ TEM sample preparation with a nanomanipulator probe is considered by many in the field to be impossible. For example, MoberlyChan, W., Marko, M. & Hsieh, C. (2005). Cryo-FIB for thinning cryo-TEM samples and evading ice during cryo-transfer. Microsc Microanal 11, Suppl 2, writes, “The ultimate may be to FIB-section into a large block of material and then “lift-out” a cryo-section for the TEM. However, “lift-out” methods remain difficult.” The reasons are explained further by Hayles M., Winter, D., Schneijdenberg, C., Meeldijk, J., Luecken, U., Persoon, H., Water, J., Jong, F., Humbel, B., and Verkleij, A. (2010). The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy. J. Struct. Biol. 172, 180-190: “ . . . the main technique of ‘lift-out’ cannot be used under cryogenic conditions due to two facts: the lift-out device, which is usually an ultra sharp needle, cannot support the vitreous temperature the sample needs and secondly the process of welding the needle to the sample using the gas injection system (GIS) will not work at cryogenic temperature well enough for such fine welds to be made.” Alexander Rigort, Felix J. B. Bäuerleinl, Elizabeth Villa, Matthias Eibauer, Tim Laugks, Wolfgang Baumeister, and Jürgen M. Plitzko (2012). Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. PNAS 109, no 12, 4449-4454, states that “Approaches such as used in the materials science (see e.g., ref 14), namely to prepare thin electron transparent windows, lift them out, and place them on EM grids, are difficult if not impossible to realize with samples which must be kept below the devitrification temperature (<−140° C.) at all times.”